Phage display has been intensively investigated for producing combinatorial antibody libraries and for presentation of combinatorial arrays of peptide elements. See, for example, Rodi et al, Curr. Opin. Biotechnol., 10:87–93, 1999; Vaughan et al, Nat. Biotechnol., 16:535–539, 1998; Griffiths et al, Curr. Opin. Biotechnol., 9:102–108, 1998; Zwick et al, Curr. Opin. Biotechnol., 9:427–436, 1998; Dall'Acqua et al, Curr. Opin. Struct. Biol., 8:443–450, 1998; Raag et al, Faseb J., 9:73–80, 1995; Barbas et al, Proc. Natl. Acad. Sci. USA, 88:7978–7982, 1991; Kang et al, Proc. Natl. Acad. Sci. USA, 88:4363–4366, 1991; Huse et al, Science, 246:1273–1278, 1989).
However, many details of the phage particle itself have not been fully elucidated and the possibility of alternative display formats also remain to be explored. The filamentous bacteriophage fd, and similarly M13, consists of a circular, single-stranded DNA molecule surrounded by a cylinder of coat proteins (FIG. 1). The molecular mass of a particle is about 1.6×107 Da of which 88% is protein and 12% is DNA (Berkowitz et al, J. Mol. Biol., 102:531–547, 1976). There are about 2700 molecules of the major coat protein pVIII that envelope the phage. At one end of the particle, there are five copies each of pIII and pVI that are involved in host-cell binding and in the termination of the assembly process. The other end contains five copies each of pVII and pIX that are hydrophobic peptides of 33 and 32 amino acids, respectively, required for the initiation of assembly and for maintenance of virion stability. While pIII, pVI, and pVIII have been used to display biological molecules, pVII and pIX have not been utilized (Rodi et al, Curr. Opin. Biotechnol., 10:87–93, 1999; Russel et al, J. Virol., 63:3284–3295, 1989).
Attempts at phage assembly in the absence of pVII and pIX almost completely abolished the production of phage. In addition, prior attempts at displaying a fusion protein on pVII or pIX previously showed that pVII and pIX were not functional with another protein fused to their N-termini (Endemann et al, J. Mol. Biol., 250:496–506, 1995), indicating that display would not be feasible using pVII, pIX, or both.
Despite the enormous attention focused on pIII- and pVIII-mediated phage display, there are no descriptions of the use of pVII or pIX for display of foreign proteins, polypeptides or antigen binding molecules, such as single chain antibodies or components of a heterodimeric protein complex.